Biallelic Knockout Research Articles

Generation of GGTA1 biallelic knockout pigs via zinc-finger nucleases and somatic cell nuclear transfer

Genetically modified pigs are valuable models of human disease and donors of xenotransplanted organs. Conventional gene targeting in pig somatic cells is extremely inefficient. Zinc-finger nuclease (ZFN) technology has been shown to be a powerful tool for efficiently inducing mutations in the genome. However, ZFN-mediated targeting in pigs has rarely been achieved. Here, we used ZFNs to knock out the porcine α-1, 3-galactosyl-transferase (GGTA1) gene, which generates Gal epitopes that trigger hyperacute immune rejection in pig-to-human transplantation. Primary pig fibroblasts were transfected with ZFNs targeting the coding region of GGTA1. Eighteen mono-allelic and four biallelic knockout cell clones were obtained after drug selection with efficiencies of 23.4% and 5.2%, respectively. The biallelic cells were used to produce cloned pigs via somatic cell nuclear transfer (SCNT). Three GGTA1 null piglets were born, and one knockout primary fibroblast cell line was established from a cloned fetus. Gal epitopes on GGTA1 null pig cells were completely eliminated from the cell membrane. Functionally, GGTA1 knockout cells were protected from complement-mediated immune attacks when incubated with human serum. This study demonstrated that ZFN is an efficient tool in creating gene-modified pigs. GGTA1 null pigs and GGTA1 null fetal fibroblasts would benefit research and pig-to-human transplantation.

Generation of multi-gene knockout rabbits using the Cas9/gRNA system.

The prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR)-associated system (Cas) is a simple, robust and efficient technique for gene targeting in model organisms such as zebrafish, mice and rats. In this report, we applied CRISPR technology to rabbits by microinjection of Cas9 mRNA and guided RNA (gRNA) into the cytoplasm of pronuclear-stage embryos. We achieved biallelic gene knockout (KO) rabbits by injection of 1 gene (IL2rg) or 2 gene (IL2rg and RAG1) Cas9 mRNA and gRNA with an efficiency of 100%. We also tested the efficiency of multiple gene KOs in early rabbit embryos and found that the efficiency of simultaneous gene mutation on target sites is as high as 100% for 3 genes (IL2rg, RAG1 and RAG2) and 33.3% for 5 genes (IL2rg, RAG1, RAG2, TIKI1 and ALB). Our results demonstrate that the Cas9/gRNA system is a highly efficient and fast tool not only for single-gene editing but also for multi-gene editing in rabbits.Electronic supplementary materialThe online version of this article (doi:10.1186/2045-9769-3-12) contains supplementary material, which is available to authorized users.

Highly Efficient Generation of GGTA1 Biallelic Knockout Inbred Mini-Pigs with TALENs

Inbred mini-pigs are ideal organ donors for future human xenotransplantations because of their clear genetic background, high homozygosity, and high inbreeding endurance. In this study, we chose fibroblast cells from a highly inbred pig line called Banna mini-pig inbred line (BMI) as donor nuclei for nuclear transfer, combining with transcription activator-like effector nucleases (TALENs) and successfully generated α-1,3-galactosyltransferase (GGTA1) gene biallelic knockout (KO) pigs. To validate the efficiency of TALEN vectors, in vitro-transcribed TALEN mRNAs were microinjected into one-cell stage parthenogenetically activated porcine embryos. The efficiency of indel mutations at the GGTA1-targeting loci was as high as 73.1% (19/26) among the parthenogenetic blastocysts. TALENs were co-transfected into porcine fetal fibroblasts of BMI with a plasmid containing neomycin gene. The targeting efficiency reached 89.5% (187/209) among the survived cell clones after a 10 d selection. More remarkably 27.8% (58/209) of colonies were biallelic KO. Five fibroblast cell lines with biallelic KO were chosen as nuclear donors for somatic cell nuclear transfer (SCNT). Three miniature piglets with biallelic mutations of the GGTA1 gene were achieved. Gal epitopes on the surface of cells from all the three biallelic KO piglets were completely absent. The fibroblasts from the GGTA1 null piglets were more resistant to lysis by pooled complement-preserved normal human serum than those from wild-type pigs. These results indicate that a combination of TALENs technology with SCNT can generate biallelic KO pigs directly with high efficiency. The GGTA1 null piglets with inbred features created in this study can provide a new organ source for xenotransplantation research.

Production of biallelic CMP-Neu5Ac hydroxylase knock-out pigs

After the knock-out (KO) of α1,3 galactosyltransfease (Gal-T), the Hanganutziu-Deicher antigen became a major antigen of the "non-Gal antigen" that is implicated in subsequent xenograft rejection. For deletion of non-Gal antigen, we successfully produced zinc finger nuclease (ZFN)-mediated monoallelic/biallelic male and female CMP-N-acetylneuraminic acid hydroxylase (CMAH) KO miniature pigs: the efficiency of the gene targeting (41.7%) was higher when donor DNA was used with the ZFN than those of ZFN alone (9.1%). Monoallelic KO pigs had no integration of exogenous DNA into their genome, indicating that this technique would provide a new avenue to reduce the risk of antibiotics resistance when organs from genetically modified pigs are transplanted into patients. Until now, both monoallelic and biallelic CMAH KO pigs are healthy and show no sign of abnormality and off-target mutations. Therefore, these CMAH null pigs on the Gal-T KO background could serve as an important model for the xenotransplantation.

Conditional Deletion of the Pten Gene in the Mouse Prostate Induces Prostatic Intraepithelial Neoplasms at Early Ages but a Slow Progression to Prostate Tumors

The PTEN tumor suppressor gene is frequently inactivated in human prostate cancer. Using Osr1 (odd skipped related 1)-Cre mice, we generated a novel conditional Pten knockout mouse strain, PtenLoxP:Osr1-Cre. Conditional biallelic and monoallelic Pten knockout mice were viable. Deletion of Pten expression was detected in the prostate of PtenLoxP/LoxP:Osr1-Cre mice as early as 2 weeks of age. Intriguingly, PtenLoxP/LoxP:Osr1-Cre mice develop high-grade prostatic intraepithelial neoplasms (PINs) with high penetrance as early as one-month of age, and locally invasive prostatic tumors after 12-months of age. PtenLoxP/+:Osr1-Cre mice show only mild oncogenic changes after 8-weeks of age. Castration of PtenLoxP/LoxP:Osr1-Cre mice shows no significant regression of prostate tumors, although a shift of androgen receptor (AR) staining from the nuclei to cytoplasm is observed in Pten null tumor cells of castrated mice. Enhanced Akt activity is observed in Pten null tumor cells of castrated PtenLoxP/LoxP:Osr1-Cre. This study provides a novel mouse model that can be used to investigate a primary role of Pten in initiating oncogenic transformation in the prostate and to examine other genetic and epigenetic changes that are required for tumor progression in the mouse prostate.

Zink finger nucleases and siRNAs: use in transgenic pig production for xenotransplantation

Emerging technologies, including the use of specific nucleases, transposons and specific siRNAs will bring significant improvements with regard to efficiency, precision and safety of gene transfer. Here, we summarize recent findings from our laboratory on the integration of Zinc‐finger nuclease technology (ZFNs) and specific siRNAs into a research programme towards the development and characterization of multi‐transgenic pigs suitable for xenotransplantation.Zinc‐finger nucleases (ZFNs) are powerful tools for producing gene knockouts (KOs) with very high efficiency. ZFNs were used to induce a biallelic knockout of the porcine α1,3‐galactosyltransferase (GGTA1) gene by employing primary porcine fibroblasts that were treated with ZFNs designed against the region coding for the catalytic core of GGTA1 in SCNT. This resulted in biallelic knockout of a high proportion of ZFN‐treated cells. ZFN‐mediated genetic modification did not interfere with the cloning process. Off‐target cleavage events or integration of the ZFN‐coding plasmid were not detected. The absence of Gal epitopes was determined by FACS and the GGTA1‐KO phenotype was confirmed by a complement lysis assay that demonstrated significant protection of GGTA1‐KO fibroblasts relative to wild‐type cells. The ZFNs were functional irrespective of the sex of the donor cells, thus allowing the effective production of female and male homozygous Gal kO pigs via somatic cloning and the rapid establishment of Gal‐/‐ pig lines. These pigs will serve as genetic background upon which additional transgenes can be inserted and expressed.SiRNAs have emerged as useful tool to knockdown specific genes in complex organisms for a variety of targets and serve as an important tool in the functional analysis of specific genes. The acute vascular rejection (AVR) remains the main hurdle for long term survival of a porcine xenograft after transplantation into primates. Immunological reactions and molecular incompatibilities can lead to endothelial activation and microvascular thrombosis. Knock‐down of tissue factor (TF) could be a promising mechanism to prevent AVR. TF is a key molecule of the extrinsic coagulation pathway. It functions as cell surface receptor for coagulation factor VIIa and thereby initiates thrombin formation. Since TF knock‐out was lethal in the mouse model, we tested different siRNAs in their efficiency in a porcine cell line to knock‐down TF. Two siRNAs reduced the mRNA level of TF to <3% compared with wild type controls as determined by RT‐PCR. Subsequently, porcine fetal fibroblasts (PFF) were co‐transfected with constructs coding for one of the promising siRNAs and a DsRed vector which confers neomycin resistance and red fluorescence. Transfected cells were selected with neomycin for 14 days and observed for the occurrence of fluorescence. Integration of the siRNA vector was confirmed by PCR. Cell clones positive for TF knock‐down siRNA served as donor cells for somatic cell nuclear transfer (SCNT). Reconstructed embryos were transferred to synchronised recipient sows. Eight transfers resulted in four pregnancies. One foster animal was sacrificed on day 35 of gestation. Three fetuses were recovered that had integrated the TF knock‐down siRNA and the derived fetal cells were used for recloning and more piglets were produced. The functionality of the knock‐down is tested in a coagulation assay and in an established TNF alpha induced assay using cultured endothelial cells of knock‐down piglets in comparison with wild type counterparts. Exposure to inflammation mediators is known stimulate expression of TF and adhesion molecules in the endothelium. Our genetically modified TF transgenic pigs will be further characterized and pigs with the desired TF knockdown will combined with already existing genetic modifications to generate multi‐transgenic pigs. This model would be of great importance to prolong xenograft survival and move porcine xenografts to the pre‐clinical level.This project was funded by DFG (Ni 256/22‐3 u. 4) and the EU project “Xenome” (LSHB‐CT‐2006‐037377).

327 PRODUCTION OF TRIPLE TRANSGENIC hHO-1/GGTA-1–/–/hCD55 TRANSGENIC PIGS USING SLEEPING BEAUTY TRANSPOSITION AND ZINC-FINGER NUCLEASES

Advances in xenotransplantation (pig-to-baboon or human transplantation) require multi-transgenic pigs with a homozygous knockout (KO) of the α1,3-galactosyltransferase gene (GGTA-1, encoding for Gal-epitopes) to control the hyperacute rejection response. To achieve prolonged survival of the porcine xenograft, transgenic expression of additional immune modulatory genes on the GGTA-1–/– background is considered the solution of choice. The acute vascular rejection (AVR) is primarily caused by endothelial cell activation and leads to rejection of GGTA-1–/– pig organs. Here, we set out to produce triple transgenic pigs with high expression of the human complement regulatory gene hCD55 (human decay acceleration factor, hDAF) and the anti-apoptotic and anti-inflammatory gene human heme oxygenase-1 (hHO-1), respectively. Porcine ear fibroblasts carrying a hHO-1 transgene (Petersen et al. 2011 Xenotransplantation 18, 355–368) were transfected with zinc-finger nucleases (ZFN) targeting the GGTA-1 gene, leading to a biallelic KO of the GGTA-1 gene in ~1% of the transfected cells (Hauschild et al. 2011 Proc. Natl. Acad. Sci. USA 108, 15 010). Somatic cell nuclear transfer (SCNT) was accomplished with cells from double transgenic cell line (hHO-1/GGTA-1–/–), and one pregnancy was terminated to obtain fetal cell cultures. Fluorescence-activated cell sorting (FACS) analysis revealed that all 11 fetuses (C6F1-F11) were lacking Gal-epitopes. One pure fetal cell culture (C6F5) was used for co-transfection with the newly designed SB-CAGGS-hDAF-Puror transposon, based on the Sleeping Beauty transposon plasmid pT2/HB, together with the SB transposase 100X plasmid (both kindly provided by Dr. Zoltan Ivics). After 14 days of selection with puromycin, cell colonies were picked and expanded to obtain cells for analysis and as a backup for SCNT. The PCR amplification with hDAF-specific primers revealed that ~50% of picked colonies had integrated the transgene. Two hDAF-positive colonies were pooled and used for SCNT. One pregnancy was sacrificed and 4 hHO-1/GGTA-1–/–/hCD55 fetuses were obtained for real-time PCR analysis and subsequent use in SCNT. Real-time PCR showed elevated hCD55 expression representing a 0.9-fold (0.7- to 1.0-fold) expression of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH), while FACS analysis confirmed absence of Gal-epitopes in all 4 fetuses. Binding of human-specific anti-CD55 antibodies showed high expression of hCD55 by FACS measurement. Triple transgenic fetuses C8F2 and C8F4 showed higher expression of hCD55 by real-time PCR and by FACS analysis compared with fetuses C8F1 and C8F3. Fetuses C8F3 and C8F4 were used for re-cloning experiments to produce live offspring, which will further be characterised and used in transplantation experiments. This work underlines the importance of new genomic technologies to further improve the efficiency of the generation of transgenic pigs suitable for xenotransplantation.

Gender non-specific efficacy of ZFN mediated gene targeting in pigs

Zinc-finger nuclease (ZFN) mediated gene disruption is significantly more efficient than conventional targeting as homologous recombination (HR) and has been successfully applied in several species, including fruit fly, rats, human and the domestic pig (Bibikova et al. 2002; Urnov et al. 2005; Geurts et al. 2009; Hauschild et al. 2011). Biallelic gene knockouts (KOs) can be achieved within one step (Urnov et al. 2005). ZFNs induce a double-strand break (DSB) which is repaired by error prone non homologous end joining (NHEJ) DNA repair creating mutations which can lead to a gene KO. Recently, we have reported the biallelic KO of the porcine a1,3-galactosyltransferase (GGTA1, Gal) gene in female porcine fetal fibroblasts that were successfully used in somatic cell nuclear transfer (SCNT) and resulted in the birth of healthy piglets with a homozygous KO of GGTA1 (Hauschild et al. 2011). The GGTA1 gene encodes the Galepitopes on the porcine cell surface which primarily are responsible for the hyperacute rejection (HAR) after pig-to-human xenotransplantation. Organs of Gal-negative pigs transplanted to baboons have shown significantly improved organ survival (up to 236 days) (Mohiuddin et al. 2012). Initially, somatic cloning was only successful with female donor cells (Cibelli et al. 1998; Kato et al. 1998; Wakayama et al. 1998; Wilmut et al. 1997) and the question was raised whether or not male donor cells were equally efficient in somatic cloning as their female counterparts. The first male cloned offspring were produced from adult tale tip cells from mice (Wakayama and Yanagimachi 1999). Subsequently, it was shown that female and male donor cells were equally efficient in producing embryonic stem cells from cloned mouse blastocysts, and other factors such as donor cell type and genotype are more important with regard to cloning efficiency (Wakayama et al. 2005). Here, we demonstrate that male primary fibroblasts are compatible with a similar targeting and cloning efficiency as their female counterparts. This will facilitate routine production of GGTA1 pigs. Production of male ZFN-mediated GGTA1 KO cells was accomplished as described before (Hauschild et al. 2011). Biallelic GGTA1-KO rate (0.8 %) was comparable to the rate obtained in female porcine cells (1 %). SCNT using male Gal-negative cells resulted in five live GGTA1 piglets and five stillJ. Hauschild-Quintern B. Petersen (&) A.-L. Queisser A. Lucas-Hahn H. Niemann (&) Institute of Farm Animal Genetics, Friedrich-LoefflerInstitut, Hoeltystrasse 10, Mariensee, 31535 Neustadt a. Rbge., Germany e-mail: bjoern.petersen@fli.bund.de

Somatic Cell Nuclear Transfer Cloning: Practical Applications and Current Legislation

Somatic cloning is emerging as a new biotechnology by which the opportunities arising from the advances in molecular genetics and genome analysis can be implemented in animal breeding. Significant improvements have been made in SCNT protocols in the past years which now allow to embarking on practical applications. The main areas of application of SCNT are: Reproductive cloning, therapeutic cloning and basic research. A great application potential of SCNT based cloning is the production of genetically modified (transgenic) animals. Somatic cell nuclear transfer based transgenic animal production has significant advances over the previously employed microinjection of foreign DNA into pronuclei of zygotes. This cell based transgenesis is compatible with gene targeting and allows both, the addition of a specific gene and the deletion of an endogenous gene. Efficient transgenic animal production provides numerous opportunities for agriculture and biomedicine. Regulatory agencies around the world have agreed that food derived from cloned animals and their offspring is safe and there is no scientific basis for questioning this. Commercial application of somatic cloning within the EU is via the Novel Food regulation EC No. 258/97. Somatic cloning raises novel questions regarding the ethical and moral status of animals and their welfare which has prompted a controversial discussion in Europe which has not yet been resolved.

Biallelic knockout of the α-1,3 galactosyltransferase gene in porcine liver-derived cells using zinc finger nucleases

BackgroundGenetic modification of the pig has been hampered by inefficiency of homologous recombination and unavailability of pig embryonic stem cells. Engineered zinc finger nuclease (ZFN)–mediated genetic modification in somatic cells combined with somatic cell nuclear transfer (SCNT) technology provides a new approach for targeted modification in pig genome. In this study, we used a ZFN pair to disrupt porcine α-1,3, galactosyltransferase (GGTA1) gene in liver-derived cells (LDC). ZFN-treated LDC were used as nuclear donors to produce fetuses and piglets via SCNT. All cloned fetuses and piglets showed biallelic knockout of GGTA1 gene. Materials and methodsA ZFN pair was designed to target exon 8 of pig GGTA1 gene. LDC were transfected with GGTA1 ZFN plasmids. SURVEYOR assay was used to evaluate the ZFN activity in LDC. GGTA1 gene knockout cells (GTKO) were obtained by counter-selection and used as nuclear donors for SCNT. The cloned fetuses and piglets were characterized by DNA sequencing. Expression of α-Gal epitope was further examined by flow cytometry and confocal microscopy. ResultsSURVEYOR assay revealed 6.48% ZFN activity in LDC. GTKO cells were obtained by counter-selection 10 d after ZFN transfection. A total of six fetuses and 13 piglets were produced by SCNT. All fetuses and piglets had biallelic mutations in the ZFN targeted region and were negative for α-Gal epitope. ConclusionsBiallelic GGTA1 gene disruption in LDC was generated efficiently by ZFN. GTKO fetuses were produced from ZFN-treated LDC by SCNT. GTKO piglets were obtained by SCNT of ZFN-treated LDC or recloning of fetal fibroblasts from GTKO fetuses. With longer lifespan and robust growth rate, LDC has the potential to endure multiple genetic modifications in vitro without going to SCNT, which could accelerate the production of genetically modified pig organs for xenotransplantation.

GRPling with PTEN

Regulation of the Pten tumor suppressor is complex and mediated by varied mechanisms. In this issue of Blood , Wey and colleagues show in a biallelic conditional knockout mouse model of GRP78 and PTEN that heterozygous loss of Grp78 suppresses leukemogenesis mediated by Pten .[1][1] These findings

Inducible knockout of GRP78/BiP in the hematopoietic system suppresses Pten-null leukemogenesis and AKT oncogenic signaling

AbstractTraditionally, GRP78 is regarded as protective against hypoxia and nutrient starvation prevalent in the microenvironment of solid tumors; thus, its role in the development of hematologic malignancies remains to be determined. To directly elucidate the requirement of GRP78 in leukemogenesis, we created a biallelic conditional knockout mouse model of GRP78 and PTEN in the hematopoietic system. Strikingly, heterozygous knockdown of GRP78 in PTEN null mice is sufficient to restore the hematopoietic stem cell population back to the normal percentage and suppress leukemic blast cell expansion. AKT/mTOR activation in PTEN null BM cells is potently inhibited by Grp78 heterozygosity, corresponding with suppression of the PI3K/AKT pathway by GRP78 knockdown in leukemia cell lines. This is the first demonstration that GRP78 is a critical effector of leukemia progression, at least in part through regulation of oncogenic PI3K/AKT signaling. In agreement with PI3K/AKT as an effector for cytosine arabinoside resistance in acute myeloid leukemia, overexpression of GRP78 renders human leukemic cells more resistant to cytosine arabinoside-induced apoptosis, whereas knockdown of GRP78 sensitizes them. These, coupled with the emerging association of elevated GRP78 expression in leukemic blasts of adult patients and early relapse in childhood leukemia, suggest that GRP78 is a novel therapeutic target for leukemia.

Targeting DNA With Fingers and TALENs.

Researchers have long recognized that the random introduction of new DNA into the genome could result in unpredictable genetic effects due to bilateral influences between transgenic and endogenous sequences. Hence, precise editing or replacement of mutant genes has been a major goal of gene therapy since its inception. That goal looked possible with the publication of studies demonstrating that targeted cleavage of chromosomal sequences and enhanced homologous recombination (HR) could be achieved using chimeric molecules composed of a nuclease domain and separate, designer DNA-recognition domains.1,2 Zinc-finger nucleases (ZFNs) are artificial endonucleases that consist of a FokI cleavage domain tethered to engineered Cys2His2 zinc-finger, DNA-binding polypeptides.3,4,5 ZFNs have gained considerable momentum and are widely considered the most mature nuclease technology in the gene therapy field.6 However, after 15 years in the spotlight, a new chimeric nuclease has emerged, Transcription Activator-Like (TAL) Effector Nucleases (TALENs).7,8,9,10 Here, we describe functional and design characteristics of ZFNs and TALENs and discuss their expanding role as tools for research and gene therapy.

233 GENDER-UNSPECIFIC KNOCKOUT OF THE GGTA1 GENE IN PIGS USING ZINC FINGER NUCLEASES

A knockout (KO) of the porcine α1,3-galactosyltransferase (GGTA1) gene is crucial for controlling the hyperacute rejection after pig-to-human xenotransplantation. Porcine kidney and cardiac xenografts from Gal-KO pigs showed prolonged survival after transplantation into baboons. Unfortunately, knockouts produced by conventional targeting (homologous recombination) are rare events and normally do not lead to biallelic KO. Zinc-finger nucleases (ZFN) have been shown to be much more efficient by inducing mutations via specific cleavage followed by nonhomologous end joining (NHEJ). Zinc-finger nucleases do not require antibiotic selection. Here, we used designed ZFN to specifically target exon 9 of the GGTA1 gene encoding the catalytic domain of the Gal-transferase. Recently, we generated female pigs with a GGTA1-KO using ZFN (Hauschild et al. 2011 PNAS 108, 12 013–12 017). Here, we investigated whether cells of a male cell line are susceptible to ZFN-mediated genome editing in a comparable manner. Male porcine fetal fibroblasts (3 × 106) were co-transfected with a ZFN-plasmid pair (7.5 μg each) by electroporation at 250 V and 400 μF. One week after transfection, a Cel-I assay revealed a NHEJ rate of 5.7% of all alleles in the cell population. After magnetic bead selection, Gal-expression was analysed by fluorescence-activated cell sorting (FACS) using fluorescein isothiocyanate (FITC)-conjugated isolectin-B4. Ninety-five percent of the cells were free of Gal epitopes, indicating a biallelic KO. These Gal-negative cells served as donor cells in somatic cell nuclear transfer (SCNT). In total, 507 transgenic embryos were transferred into 6 recipient sows. By obtaining live animals by SCNT after transfer of male ZFN-GGTA1-KO embryos, we will have produced female and male ZFN-KO pigs, which can be used for further breeding experiments to circumvent the extensive and relative inefficient recloning method. These results show that ZFN work independent of the sex of the cells and that a biallelic Gal-KO can be produced in male cells by using the ZFN technology. This technology could benefit both agriculture and biomedicine and establishes the pig as a model for human diseases.

Correction for Hauschild et al., Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases

Correction for Hauschild et al., Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases

Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases

Zinc-finger nucleases (ZFNs) are powerful tools for producing gene knockouts (KOs) with high efficiency. Whereas ZFN-mediated gene disruption has been demonstrated in laboratory animals such as mice, rats, and fruit flies, ZFNs have not been used to disrupt an endogenous gene in any large domestic species. Here we used ZFNs to induce a biallelic knockout of the porcine α1,3-galactosyltransferase (GGTA1) gene. Primary porcine fibroblasts were treated with ZFNs designed against the region coding for the catalytic core of GGTA1, resulting in biallelic knockout of ∼1% of ZFN-treated cells. A galactose (Gal) epitope counter-selected population of these cells was used in somatic cell nuclear transfer (SCNT). Of the resulting six fetuses, all completely lacked Gal epitopes and were phenotypically indistinguishable from the starting donor cell population, illustrating that ZFN-mediated genetic modification did not interfere with the cloning process. Neither off-target cleavage events nor integration of the ZFN-coding plasmid was detected. The GGTA1-KO phenotype was confirmed by a complement lysis assay that demonstrated protection of GGTA1-KO fibroblasts relative to wild-type cells. Cells from GGTA1-KO fetuses and pooled, transfected cells were used to produce live offspring via SCNT. This study reports the production of cloned pigs carrying a biallelic ZFN-induced knockout of an endogenous gene. These findings open a unique avenue toward the creation of gene KO pigs, which could benefit both agriculture and biomedicine.

Abstract LB-37: Inducible knockout of GRP78/BiP in the hematopoietic system suppresses Pten-null leukemogenesis and AKT oncogenic signaling

Abstract Traditionally GRP78 is regarded as protective against hypoxia and nutrient starvation prevalent in the microenvironment of solid tumors, thus, its role in hematologic malignancies remains to be determined. In this study, elevated GRP78 expression was detected in leukemic blasts of adult patients, leukemia cell lines and inversely correlates with time to relapse in childhood acute lymphocytic leukemia. To directly elucidate the requirement of GRP78 in leukemogenesis, we created a biallelic conditional knockout mouse model of GRP78 and PTEN in the hematopoietic system. Strikingly, heterozygous knockdown of GRP78 in PTEN null mice is sufficient to restore the hematopoietic stem cell (HSC) population back to the normal percentage and suppress leukemic blast cell expansion. AKT/mTOR activation in PTEN null bone marrow cells is potently inhibited by Grp78 heterozygosity, corresponding with suppression of AKT phosphorylation by GRP78 knockdown in leukemia cell lines. This is the first demonstration that GRP78 is a critical effector of leukemia progression, at least in part through control of oncogenic AKT/mTOR signaling. Furthermore, overexpression of GRP78 renders human leukemic cells more resistant to AraC-induced apoptosis whereas knockdown of GRP78 sensitizes them, suggesting GRP78 is a novel potent therapeutic target for leukemia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-37. doi:10.1158/1538-7445.AM2011-LB-37

Abstract SY06-01: Critical role of the stress chaperone and cell signaling regulator GRP78 in cancer

Abstract SY06-01: Critical role of the stress chaperone and cell signaling regulator GRP78 in cancer

329 GENETIC TARGETING OF THE PORCINE α1,3-GALACTOSYLTRANSFERASE GENE IN FETAL FIBROBLAST CELLS USING ZINC FINGER NUCLEASES

Hyperacute rejection after porcine-to-human xenotransplantation is caused by binding of preformed human antibodies against Gal-epitopes on the surface of porcine cells. Organs from Gal-negative pigs have shown prolonged survival after transplantation into baboons. Knocking out a gene by conventional gene targeting frequency is extremely inefficient (homologous recombination = 0.0001 to 0.001%; Denning et al. 2001). Recent publications in rats (Geurts et al. 2009) show that the gene knockout via zinc finger nuclease (ZFN)-driven nonhomologous end joining (NHEJ) can be enhanced 10 000-fold over conventional approaches, making it feasible to generate a biallelic gene knockout with one ZFN application. Here, we used ZFN technology to generate porcine cells that carry a ZFN-mediated knockout of the Gal gene to use these cells as donor cells in somatic cell nuclear transfer (SCNT) to obtain live offspring. One primary porcine fetal fibroblast cell line was transfected by electroporation (n = 6) with a pair of ZFN plasmids designed to target the DNA sequence encoding the catalytic domain located in exon 9 of the α1,3-gal locus. Transfected cells were incubated (7 days at a combination of 30°C and 37°C) and analysed for Gal expression by fluorescence activated cell sorting (FACS) using fluorescein isothiocyanate (FITC)-conjugated isolectin-B4. On average, 1.4% (± 0.3%; n = 6) of the cells were free of Gal epitopes, indicating a biallelic knockout. DNA mutation detection analysis (Cel-I assay) of cell cultures gave a mean frequency of 3.5% NHEJ (± 1.3%; n = 6) giving the fraction of mutant alleles within the cell population. One cell line with 1% Gal-negative cells was sorted by a magnetic Dynabead-based separation method to select for Gal-negative cells (Fujimura et al. 2008). Because of the limited amount of Gal-negative cells within the cell population, we chose to select the cells with magnetic beads. This method is gentler to the cells and leads to a higher plating efficiency after sorting compared with FACS. The sorted cells could be easily expanded and will serve as donor cells in SCNT to show the feasibility of generating knockout pigs via ZFN-mediated gene knockout. This study demonstrates that ZFN technology is an applicable tool to produce genetically modified porcine cells for use as donors in SCNT and to speed the creation of pig models for xenotransplantation and human diseases.

Generation of a triple‐gene knockout mammalian cell line using engineered zinc‐finger nucleases

Mammalian cells with multi-gene knockouts could be of considerable utility in research, drug discovery, and cell-based therapeutics. However, existing methods for targeted gene deletion require sequential rounds of homologous recombination and drug selection to isolate rare desired events--a process sufficiently laborious to limit application to individual loci. Here we present a solution to this problem. Firstly, we report the development of zinc-finger nucleases (ZFNs) targeted to cleave three independent genes with known null phenotypes. Mammalian cells exposed to each ZFN pair in turn resulted in the generation of cell lines harboring single, double, and triple gene knockouts, that is, the successful disruption of two, four, and six alleles. All three biallelic knockout events were obtained at frequencies of >1% without the use of selection, displayed the expected knockout phenotype(s), and harbored DNA mutations centered at the ZFN binding sites. These data demonstrate the utility of ZFNs in multi-locus genome engineering.